Understanding the function and significance of microRNAs (miRNAs or miRs) in the regulation of xenobiotic disposition and actions is important for developing new rational therapies. However, there is a lack of efficient means to produce large quantities of natural, biologically active miRNA agents for prospective mechanistic and therapeutic studies in vivo. To fill this gap, the PI's lab has investigated the feasibility of producing recombinant pre-miRNA (mir) agents, which may be processed to functional mature miRNAs in human cells. These studies have led the PI to develop a novel pre-miRNA-based optimal noncoding RNA scaffold (OnRS) for achieving a consistent, high-yield, and large-scale expression of recombinant OnRS/miRNAs and OnRS/siRNAs in a common strain of E. coli. After purification and structural characterization, recombinant ncRNAs were found to be selectively processed to target miRNAs (or siRNAs) within various human cell line systems. Consequently, recombinant miRNAs reduced target gene expression, altered cellular xenobiotic metabolism capacity, and sensitized the cells to small-molecule drugs. Moreover, in vivo-jetPEI-formulated recombinant ncRNAs were well tolerated in mouse models and suppressed target gene expression in vivo. Given these exciting preliminary findings, we hypothesize that biologically active miRNA agents (e.g., miR-27b, miR-34a, and miR-124) can be engineered on a large scale using this novel OnRS-based recombinant RNA technique, and the OnRS/miRNAs may serve as unique tools to define the importance of miRNAs in the modulation of drug disposition and efficacy. To test this hypothesis, we will refine and standardize OnRS technique, produce a set of OnRS/miRNA and OnRS/siRNA agents, and define their primary structural characteristics including posttranscriptional modifications (Aim 1), delineate the mechanistic functions of OnRS/miRNAs in the control of cellular xenobiotic metabolism and disposition in vitro (Aim 2), and establish the efficacy of OnRS/miRNAs in the modulation of pharmacokinetics and therapeutic outcomes in clinically relevant animal models in vivo (Aim 3). This research shall establish a novel OnRS-based recombinant RNA technique and open new avenues to miRNA pharmacoepigenetics.